The long-term objective of this work is to compare the genetic maps of eutherian mammals (human and mouse) with those of metatherian (marsupial) and prototherian (monotreme) mammals in order to gain a better understanding of the nature and extent of chromosome rearrangement involved in the evolution of these modern species from a common ancestor. One specific aim is to prepare radiolabeled or biotinylated cloned probes that have been mapped to specific sites throughout the length of human chromosome 3 and to map these by standard or fluorescence in situ hybridization to metaphase chromosomes of a marsupial and a monotreme. Do they map to a single chromosome or to multiple chromosomes in the marsupial or in the monotreme? For those that are still linked, is their order conserved or altered? How large a cluster of genes shows a conserved linkage or a conserved order of linked loci? Are there regional differences, such as greater conservation of loci near a centromere or greater variability of loci near a telomere? Another specific aim is to characterize the 20-25% of the genome present as highly repetitive sequence DNA in the centromeric regions of marsupial and monotreme chromosomes using molecular methods in addition to cytologic methods involving in situ hybridization, restriction endonuclease banding, and antibodies to 5-methylcytosine. The dual aim of these studies is to construct a beginning physical map of the 20-25% of the genome present as centromeric heterochromatin and to identify the repetitive sequences that are closest to, or within, the centromere as a first step in characterizing sequences that may be involved in centromeric function in these mammals as an extension of our ongoing studies of the centromere in human, mouse and rat.